Suppression of uPA and uPAR Attenuates Angiogenin Mediated Angiogenesis in Endothelial and Glioblastoma Cell Lines.pdf (2.5 MB)
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Suppression of uPA and uPAR Attenuates Angiogenin Mediated Angiogenesis in Endothelial and Glioblastoma Cell Lines

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posted on 27.05.2011, 00:00 by Hari Raghu, Sajani S. Lakka, Christopher S. Gondi, Sanjeeva Mohanam, Dzung H. Dinh, Meena Gujrati, Jasti S. Rao
Background: In our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood. Methodology/Principal Findings: In the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells. Conclusions/Significance: Taken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway.

Funding

This research was supported by a grant from National Institutes of Health (NIH), CA 75557 (to J.S.R.).

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Publisher Statement

© 2010 Raghu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The original source for this publication is at the Public Library of Science. DOI: 10.1371/journal.pone.0012458.

Publisher

Public Library of Science

Language

en_US

issn

1923-6203

Issue date

27/08/2010

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